Functional diversification of sonic hedgehog paralog enhancers identified by phylogenomic reconstruction

Investigation of the ar-C midline enhancer of sonic hedgehog orthologs and paralogs from distantly related vertebrate lineages identified lineage-specific motif changes; exchanging motifs between paralog enhancers resulted in the reversal of enhancer specificity

Shadow enhancers: Frequently asked questions about distributed cis ‐regulatory information and enhancer redundancy

This paper, in the form of a frequently asked questions page (FAQ), addresses outstanding questions about “shadow enhancers”, quasi‐redundant cis ‐regulatory elements, and their proposed roles in transcriptional control. Questions include: What exactly are shadow enhancers? How many genes have shadow/redundant/distributed enhancers? How redundant are these elements? What is the function of distributed enhancers? How modular are enhancers? Is it useful to study a single enhancer in isolation? In addition, a revised definition of “shadow enhancers” is proposed, and possible mechanisms of shadow enhancer function and evolution are discussed. The term “shadow enhancer” was coined rather recently and this article not only gives a revised definition of this term but also discusses possible mechanisms of shadow enhancer function and evolution.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90151/1/135_ftp.pd

A Multicassette Gateway Vector Set for High Throughput and Comparative Analyses in Ciona and Vertebrate Embryos

BACKGROUND: The past few years have seen a vast increase in the amount of genomic data available for a growing number of taxa, including sets of full length cDNA clones and cis-regulatory sequences. Large scale cross-species comparisons of protein function and cis-regulatory sequences may help to understand the emergence of specific traits during evolution. PRINCIPAL FINDINGS: To facilitate such comparisons, we developed a Gateway compatible vector set, which can be used to systematically dissect cis-regulatory sequences, and overexpress wild type or tagged proteins in a variety of chordate systems. It was developed and first characterised in the embryos of the ascidian Ciona intestinalis, in which large scale analyses are easier to perform than in vertebrates, owing to the very efficient embryo electroporation protocol available in this organism. Its use was then extended to fish embryos and cultured mammalian cells. CONCLUSION: This versatile vector set opens the way to the mid- to large-scale comparative analyses of protein function and cis-regulatory sequences across chordate evolution. A complete user manual is provided as supplemental material

Targeted transgene integration overcomes variability of position effects in zebrafish.

Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish.This work was funded by ‘BOLD’ Marie-Curie Initial Training Network; and ‘ZFHealth’ Integrating project in the Framework 7 programme of the European RESEARCH ARTICLE Development (2014) doi:10.1242/dev.100347Development Commission; University of Birmingham (F.M.); Temple University; and the National Institutes of Health (NIH) [HD061749 to D.B.]

From Royal Toleration to Republican Equality : a Comparative Approach to Religious Liberty in France and America from 1663 to 1833

Au XVIIe siècle, en France et en Angleterre, on parlait de tolérance ou d’indulgence lorsque l’on permettait à d’autres croyances que celle du souverain de se manifester. Ce vocabulaire fut remis en question avec l’avènement de la liberté, qui se trouve être indépendante du bon vouloir d’un prince. Plus encore, le discours des révolutions du XVIIIe siècle affirma l’égalité des droits qui, davantage que de protéger la liberté de culte, interdit le traitement particulier d’un citoyen du fait de sa religion. Un certain nombre d’inerties idéologiques se manifestèrent au cours de cette progression, mais aussi autant de contradictions, qui montrent qu’un apprentissage fut nécessaire. Nous décrivons ce processus graduel, qui mena de la tolérance royale à l’égalité républicaine. Nous en étudions la trajectoire aux États-Unis, et nous comparons cette évolution avec celle que connut la France, car il apparaît que le droit français présente des similitudes avec le modèle américain. Aussi, la Révolution française a marqué les esprits en Amérique, notamment par les violences commises envers le clergé, qui accompagnèrent la construction républicaine. Cette approche comparative permet d’interroger, dans le champ historique, la théorie de « la civilisation atlantique » et son éventuelle application au domaine de la liberté religieuse. Par ailleurs, dans le champ civilisationnel, il nous devient possible d’identifier des éléments proprement américains dans le modèle de séparation entre État et Églises. Les chartes, constitutions et déclarations des droits constituent l’essentiel de notre corpus. Il peut être borné symboliquement de 1663 (Charte royale du Rhode Island) à 1833 (11e amendement à la Constitution du Massachusetts), pour jalonner l’évolution de la relation entre État et Églises. Pour expliciter ces écrits il nous faudra tenir compte du contexte ecclésial et doctrinal protestant en Amérique. Il participa à cette construction du droit, et concourut aussi à maintenir un décalage avec le modèle français. Enfin, le recours à des sources anglophones permettra d’interroger le sens particulier d’un vocabulaire de la liberté religieuse, dont la transparence sémantique doit être relativisée, dans l’espace et dans le temps.In the 17th century, in France and England, free expression of any religious persuasion dissenting from that of the sovereign was referred to toleration or indulgence. Such words were to be questioned with the advent of the term liberty, which is independent from a prince’s goodwill. Furthermore, discourse from the revolutions in the 18th century introduced the notion of equality of rights which went beyond protecting liberty of worship to forbid any unequal treatment on account of religion. This form of progress encountered some ideological resistance and many contradictions, proof that a learning process was at work. This dissertation analyzes the gradual process that led from royal toleration to republican liberty, and compares its evolution in the United States to the one in France as it appears that French law may have been inspired by the American model. The French Revolution also made a deep impression in America, especially with the outbreak of violence against the clergy that accompanied republican development. From a historical point of view, this comparative approach helps determine if the Atlantic revolution theory may be applied to the question of religious liberty. In addition, in the context of American studies, it will help identify some elements proper to the United States’ model of separation of Church and State. The corpus is essentially made up of charters, constitutions and declarations of rights. It can be symbolically delimited in time from 1663 (Rhode Island’s Royal Charter) to 1833 (11th Amendment to the Massachusetts Constitution) to monitor developments in relations between Church and State. Assessing the American Protestant ecclesial and doctrinal context will help clarify these documents as it played a role in the development of the law and contributed to maintain a certain distance with the French model. Finally, the recourse to English language sources has made possible the analysis of the specific meaning of vocabulary related to religious liberty, whose semantic transparency must be put into perspective, both in space and time

From Royal Toleration to Republican Equality : a Comparative Approach to Religious Liberty in France and America from 1663 to 1833

Au XVIIe siècle, en France et en Angleterre, on parlait de tolérance ou d’indulgence lorsque l’on permettait à d’autres croyances que celle du souverain de se manifester. Ce vocabulaire fut remis en question avec l’avènement de la liberté, qui se trouve être indépendante du bon vouloir d’un prince. Plus encore, le discours des révolutions du XVIIIe siècle affirma l’égalité des droits qui, davantage que de protéger la liberté de culte, interdit le traitement particulier d’un citoyen du fait de sa religion. Un certain nombre d’inerties idéologiques se manifestèrent au cours de cette progression, mais aussi autant de contradictions, qui montrent qu’un apprentissage fut nécessaire. Nous décrivons ce processus graduel, qui mena de la tolérance royale à l’égalité républicaine. Nous en étudions la trajectoire aux États-Unis, et nous comparons cette évolution avec celle que connut la France, car il apparaît que le droit français présente des similitudes avec le modèle américain. Aussi, la Révolution française a marqué les esprits en Amérique, notamment par les violences commises envers le clergé, qui accompagnèrent la construction républicaine. Cette approche comparative permet d’interroger, dans le champ historique, la théorie de « la civilisation atlantique » et son éventuelle application au domaine de la liberté religieuse. Par ailleurs, dans le champ civilisationnel, il nous devient possible d’identifier des éléments proprement américains dans le modèle de séparation entre État et Églises. Les chartes, constitutions et déclarations des droits constituent l’essentiel de notre corpus. Il peut être borné symboliquement de 1663 (Charte royale du Rhode Island) à 1833 (11e amendement à la Constitution du Massachusetts), pour jalonner l’évolution de la relation entre État et Églises. Pour expliciter ces écrits il nous faudra tenir compte du contexte ecclésial et doctrinal protestant en Amérique. Il participa à cette construction du droit, et concourut aussi à maintenir un décalage avec le modèle français. Enfin, le recours à des sources anglophones permettra d’interroger le sens particulier d’un vocabulaire de la liberté religieuse, dont la transparence sémantique doit être relativisée, dans l’espace et dans le temps.In the 17th century, in France and England, free expression of any religious persuasion dissenting from that of the sovereign was referred to toleration or indulgence. Such words were to be questioned with the advent of the term liberty, which is independent from a prince’s goodwill. Furthermore, discourse from the revolutions in the 18th century introduced the notion of equality of rights which went beyond protecting liberty of worship to forbid any unequal treatment on account of religion. This form of progress encountered some ideological resistance and many contradictions, proof that a learning process was at work. This dissertation analyzes the gradual process that led from royal toleration to republican liberty, and compares its evolution in the United States to the one in France as it appears that French law may have been inspired by the American model. The French Revolution also made a deep impression in America, especially with the outbreak of violence against the clergy that accompanied republican development. From a historical point of view, this comparative approach helps determine if the Atlantic revolution theory may be applied to the question of religious liberty. In addition, in the context of American studies, it will help identify some elements proper to the United States’ model of separation of Church and State. The corpus is essentially made up of charters, constitutions and declarations of rights. It can be symbolically delimited in time from 1663 (Rhode Island’s Royal Charter) to 1833 (11th Amendment to the Massachusetts Constitution) to monitor developments in relations between Church and State. Assessing the American Protestant ecclesial and doctrinal context will help clarify these documents as it played a role in the development of the law and contributed to maintain a certain distance with the French model. Finally, the recourse to English language sources has made possible the analysis of the specific meaning of vocabulary related to religious liberty, whose semantic transparency must be put into perspective, both in space and time

Vertebrate floor-plate specification: variations on common themes

International audienceSituated at the ventral-most part of the vertebrate neural tube, the floor plate (FP) is an important signalling centre that controls the regional differentiation of neurons in the nervous system. It secretes guidance molecules that direct ventrally navigating axons crucial for the correct wiring of neuronal circuits. Although the function of the FP is well-conserved from fish to humans, discrepancies exists with respect to both the signalling system involved in FP induction, and the origin of the FP in various vertebrate species. Recent findings from the embryos of zebrafish, chicken and mouse provide insights that reconcile previous results and suggest common themes in vertebrate FP specification

Cooperation of sonic hedgehog enhancers in midline expression

International audienceIn zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution

Mutation in the δ-subunit of the nAChR suppresses the muscle defects caused by lack of dystrophin

10.1002/dvdy.20592Developmental Dynamics23441016-1025DEDY